Project Summary At this point in time, it is generally understood and agreed upon that single-molecule sequencing (SMS) is the future of genomics, transcriptomics, epigenomics, and epitranscriptomics due to its significant advantages over other technologies and methods. However, in order for these advantages to be fully realized, and for SMS to become the ?gold standard? sequencing approach, significant issues and hurdles must be solved and overcome. During this program, Electronic BioSciences, Inc. (EBS) aims to demonstrate a completely new and enabling SMS method that will possess the ability to directly and correctly identify individual nucleotides, including chemically modified nucleotides. During this project, we will both demonstrate the ability of this entirely new sequencing approach to sequence DNA with high accuracy (directly comparing the obtained accuracy, throughput, error mechanisms and associated rates to other SMS approaches) and correctly identify (and sequence) 5-methylcytosine (5mC) and its derivatives, at the single molecule level. At the conclusion of this Phase I project, we will have successfully demonstrated an entirely new and dramatically improved SMS approach, and reduced the associated risks involved with its full future commercial developments. There is a current need within the field of next generation sequencing (NGS) or so called third generation sequencing (TGS) for new, enabling instrumentation that is capable of high-accuracy, direct, native DNA sequencing, including the ability to correctly identify canonical and modified bases, homopolymer stretches, and sequence repeats. The entirely new SMS methodology that will be developed during this project will overcome known hurdles and limitations of currently available NGS, TGS, and SMS technologies, resulting in technology that is cost-efficient, highly accurate, easy to setup and utilize, capable of de novo sequencing and modified base calling, and yields highly simplistic data for easy analysis and post possessing. Through significant advancements made during this program, this resulting technology will revolutionize the use of the genome and epigenome, radically change standard R&D and clinical practices, and greatly advance clinical diagnostics, prognostics, and therapeutic decision making.